| Bactrocera dorsalis (Hendel) is a destructive insect species that can cause severe economic losses to fruit and vegetable industry. The B. dorsalis transcriptome library has provided massive useful information for the molecule research on it. Molecular targets were selected to research on their functions so as to provide theoretical basis for the prevention and control of the oriental fruit fly. Rab protein is a class of small GTP binding protein which is important for vesicular trafficking and signal transduction. Rabll is one member of the Rab family and plays many roles in the development of insects. However, the function of Rabll in the metamorphosis of holometabolous insects remains unclear.The Rab family of B. dorsalis transcriptome library was chosen as the object of study in this research. Through the research on the gene expression profiles, it is found that BdRabll may be have involved in the pupation. Molecular biological techniques like real-time fluorescence quantification PCR and RNAi were employed to study the genes expression profiles and the role of Rab in the metamorphosis of B. dorsalis. The results has not only enhanced our understanding of the B. dorsalis development mechanism, but also provided new ways and possibilities to prevent and control the insect.1. Verification differential expression genes in B. dorsalis transcriptome library.Through real-time fluorescence quantification PCR, the expression profiles of8differential expression genes which were from the B. dorsalis transcriptome library and also related to the development and reproduction were verified. In this way, the researchers evaluated the accuracy and reliability of the library. The Rab family whose expression level was very high in metamorphosis were selected as the candidate gene for further research.2. Bioinformatic analysis and expression profiles of B. dorsalis Rab family.The results of the homology comparison for the Bactrocera dorsalis Rab family showed that, Rab family amino acid sequences were highly similar to affinis insects with the highest similarity, found in BdRabll, being99%compared with Drosophila melanogaster DmRabll, and the lowest in BdRab18and BdRab40, being85%compared with Glossina morsitans Rab18and D. melanogaster DmRab40. The comparison between the amino acid sequences of B. dorsalis Rab family showed that they all had the amino acid residue locus that forms GTP/Mg2+binding sites and Rab molecular characteristics. Phylogenetic tree analysis found that the evolutionary distance of B. dorsalis Rab family were almost the same as that of the corresponding genes in D. melanogaster.Research on the expression patterns of B. dorsalis Rab family genes in different developmental stages showed that the expression pattern differs among genes. BdRabl and BdRab35had a high expression level in3rd instar larvae and pupa stage, BdRab5adult stage and BdRab7prepupa stage, while BdRabll, BdRab14and BdRab18were all notably expressed in the3rd larval instar stage and BdRab40’s high expression level appeared in3rd instar larvae and adult stage. These indicate that each gene functioned differently during the growth and development of B. dorsalis. The3rd instar larva is the preparation stage before pupation, when holometabolous insects were undergoing drastic dissociation of old tissues and the genesis of new tissues. At this period plenty of nutrition and energy were needed. Before pupation, insects would take abundant food and grow fast, and their body size grows drastically to prepare nutrition for pupation. The research showed that, BdRabll, BdRab14and BdRabl8may be involed in the preparation activities for pupation.3. The research on tissue expression profile and function of BdRabll.We chose BdRabll as the target gene to study the function in the development of B. dorsalis. BdRabll ORF was cloned with a size of642bp and encoding213amino acids. Researches on the expression patterns of BdRabll in different tissues found that the expression level of BdRabll was the highest in the fat body of3rd larval instar, and compared to2nd larval instar fat body and prepupa fat body, the expression level in3rd larval instar was significant. Interference of BdRabll gene of3rd larva instar through RNAi technology caused notable decline (P<0.01) in the expression of the gene and the gene expression of10ng/μl dsRNA treatment group declined by65.7%. The decline in BdRabll expression, on one hand, resulted in changes in the form of3rd larva instar fat body—compared with control group, the fat body form of treatment group, transparent and not full inside, was notably different; on the other hand, it affected the eclosion rate of Bactrocera dorsalis—the eclosion rate of treatment group (58.5%) was significantly (P<0.05) lower than that of the control group (85.9%and91.1%). However, the impact of the decline in BdRabll expression on the longevity of the insect was not remarkable.It is inferred that BdRabll takes part in the nutrition accumulation and storage of3rd larva instar fat body cells, thus influencing the fat body form and the eclosion of Bactrocera dorsalis. Since that Rabll protein transports substances through intracellular vesicular transport and extracellular endocytosis has been massively documented, the researcher believes that BdRabll provides and transports substances for the nutrition accumulation and storage by fat body cells. Nevertheless, the molecular mechanism of BdRabll and the specific situation of its signal path in the upstream and downstream remain unknown and have yet to be found out. |
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